Droplet digital PCR for quantification of ITGA6 in a stool mRNA assay for the detection of colorectal cancers

Abstract

Aim: To investigate the use of droplet digital polymerase chain reaction (ddPCR) for detecting host mRNA markers in stools as a non-invasive test for colorectal cancer screening.

Methods: ddPCR and quantitative PCR were compared side by side for their performance in the detection of ITGA6 and ITGA6A transcripts in stool samples obtained from patients with various types of colorectal lesions (advanced adenomas and stage II-IV colorectal cancers) and control (patients displaying no pathological findings) using duplex TaqMan reactions for both methods. ITGA6 and ITGA6A were chosen for this proof-of-concept study based on their relative medium and low abundance in stool samples, respectively, as established in a previous study.

Results: We found that the ddPCR and qPCR methods performed equally well in this TaqMan duplex assay for the detection of ITGA6 and ITGA6A transcripts in stools of patients with colorectal lesions. For ITGA6, receiver operating characteristic (ROC) curve analysis showed comparable areas under the curve of 0.91 (P < 0.0001) and 0.89-0.90 (P < 0.0001) for the prediction of advanced adenomas and colorectal cancers, respectively. ITGA6A, which was detected at very low levels in control patients, was found to be significantly elevated (over 40 times) in stage II and III colorectal cancers (P < 0.0002). Comparison of the two sets of data revealed a strong correlation of the copy numbers obtained by ddPCR and qPCR for both ITGA6 and ITGA6A.

Conclusion: We found that ITGA6 and ITGA6A detection in stools of patients with colorectal cancers with ddPCR is comparable to that of qPCR using TaqMan assays.

Keywords: Advanced adenoma; Biomarker; Colorectal cancer; Non-invasive screening; mRNA.

Figure 1

Detection of ITGA6 and ITGA6A in stool samples of controls and patients diagnosed with Ad and stage II-IV CRC by duplex ddPCR. A: For total ITGA6, significant increases in copy number were observed for Ad and all tested CRC stages relative to the controls (Ctrl); B: ROC curve analysis of ITGA6 detection in Ad and in stages II-III CRC. Area under the curve (AUC), sensitivity and specificity are provided (95%CI); C: For ITGA6A, significant increases in copy number were only observed for stage II and stage III CRC relative to Ctrl; D: Accordingly, ITGA6A/ITGA6 ratios were found to be significantly different between controls and stage II and III CRC. Results in A, C and D are expressed as median (interquartile range) of copy number relative to control patients. ^aP < 0.05, ^cP < 0.01, ^eP < 0.001 using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.

Figure 2

Detection of ITGA6 and ITGA6A in stool samples of controls and patients diagnosed with Ad and stage II-IV CRC by duplex qPCR. A: For total ITGA6, significant increases in copy number were observed for Ad and all tested CRC stages relative to Ctrl; B: ROC curve analysis of ITGA6 detection in Ad and in stages II-III CRC. AUC, sensitivity and specificity are provided (95%CI); C: For ITGA6A, significant increases in copy number were only observed for stage II and stage III CRC relative to Ctrl; D: Accordingly, ITGA6A/ITGA6 ratios were found to be significantly different between controls and stage II and III CRC. Results in A, C and D are expressed as median (interquartile range) of copy number relative to control patients. ^aP < 0.05, ^cP < 0.01, ^eP < 0.001 using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.

Figure 3

Correlation between ddPCR and qPCR measurements. ITGA6 (A) and ITGA6A (B) levels (copy number) detected in controls (Ctrl: white), Ad (red) and CRC (blue) were plotted and evaluated using the nonparametric Spearman correlation test.